The project continues the study of the interaction between nerve and muscle during synaptogenesis. A receptor aggregation factor (RAF), produced specifically by neurons, aggregates the acetylcholine receptors (AChR) of cultured embryonic myotubes. The nature of the RAF and its effects on muscle are relevant to the development of the neuromuscular junction. The mechanism of RAF action was studied. Substances known to bind to and to crosslink the AChR produce small AChR microaggregates but prevent the formation of large aggregates by RAF. When the lipid composition of the myotube membrane was changed by the addition of fatty acids, only linoleic acid increased the AChR aggregation. Various components of basement membrane alone or with RAF, had no effect on AChR distribution, except laminin, which acted synergistically with RAF to produce AChR aggregation. Further purification of RAF has shown that it is a glycoprotein. Factors derived from neurons also produce a general trophic effect on myotube cultures and decrease the rate of AChR degradation. These effects co-purify with the RAF.